There is a global need for developing safe and effective vaccines for controlling diseases. Vaccines aim to evoke immune response leading to the production of antibodies (humoral immunity) or cell-mediated responses that will counter the particular disease. An ideal vaccine elicits an effective immunogenic response with a reasonable duration of immunity, has minimal adverse side effects, is economically feasible and is relatively simple to produce and use.
The vaccination field focuses on different types of vaccines and effective means of delivery. There are many types of vaccines, including inactivated, live attenuated, recombinant vaccines such as viral and bacterial vectors, toxoids, DNA vaccines and synthetic polypeptide combined vaccines. The vaccines can be delivered orally or parenterally. The parenteral administration of vaccines is inconvenient because of the risks involved and further because of the recipient's pain and fear. In addition, when the target organisms are aquatic animals or a large number of land animals, parenteral administration is practically impossible.
Aquaculture is a constantly growing food producing sector. Disease prevention is a key issue to maintain a sustainable development of aquaculture. Optimal husbandry and general management practices including biosecurity (protection from infectious agent), nutrition, genetics, system management and water quality are critical for maximizing aquatic animal health. However, all facilities are vulnerable to disease outbreaks because many pathogenic organisms are opportunistic and present in the environment. In addition, when species are reared at high densities, agents of infectious disease are easily transmitted between individuals. Treatments hitherto mainly include administering antibiotics and vaccines. Consumer health, food safety issues and concerns regarding the development of resistant bacteria reduce the use of antibiotics in aquaculture. Moreover, viral diseases cannot be treated with available antibiotics. The development of additional fish vaccines and effective means of delivery will lead to significantly decreased use of antibiotics in aquacultures.
Streptococcus iniae is a species of Gram-positive bacterium that is a leading fish pathogen in aquaculture operations worldwide. It infects a variety of cultured and wild fish which results in severe economic loss. Control of Streptococcus iniae by vaccination has met with limited success, thus antibiotic usage is the main current practice for reducing mortality and consequent economic loss.
U.S. Pat. No. 6,379,677 discloses polyvalent vaccines against Streptococcus iniae prepared from formalin-killed cells and concentrated extracellular products of Streptococcus iniae. 
Streptococcus agalactiae is another important pathogen that affects aquatic species as well as animals and humans. It has been found worldwide in a variety of fish species, especially those living in warm water. For example, it was found in ya-fish (Schizothorax prenanti) farms in Sichuan Province, China (Geng Y. Transboundary and Emerging Diseases 59(4):369-375, 2012, Abstract). U.S. Pat. No. 7,204,993 discloses a composition comprising killed cells of isolated Streptococcus agalactiae as a vaccine for fish. The composition is suggested to be administrated by intraperitoneal or intramuscular injection, bath immersion, oral administration, or nasal administration.
The infectious salmon anemia (ISA) virus is of the family Orthomyxoviridae, genus Isavirus. ISA is a serious disease of farmed Atlantic salmon. ISA was first detected in Norway in 1984 and was characterized by severe anemia and circulatory disturbances. As with other viral vaccines for fish, commercially available vaccines against ISA, based upon inactivated whole virus, have questionable field effectiveness.
Viral nervous necrosis (VNN) is caused by nervous necrosis viruses (NNV) that are a major pathogen of several economically important fish species worldwide. The NNV are non-enveloped, small single-stranded sense RNA viruses. Betanodaviruses cause viral nervous necrosis (VNN) or viral encephalopathy and retinopathy (VER). More than 40 fish species, most of them marine, were reported to be susceptible to Betanodavirus (Nakai T. et al. The Israeli journal of aquaculture, 61(3):198-207 2009) Immunization using inactivated betanodavirus was suggested as an effective strategy to protect grouper larvae against VNN. Effective NNV vaccines must be administered at the early larval stage before infection with NNV has occurred. Due to the small size of larvae and their sensitivity to stress, an oral vaccination is a more suitable means of immunization than injection or immersion.
Salmonella infection is a major cause of gastroenteritis in humans worldwide and it is often associated with consumption of raw or uncooked poultry products. Eggs contaminated with Salmonella enterica Serovar enteritidis are associated with a significant number of human illnesses and continue to be a public health concern. A substantial reduction of the intestinal population of Salmonella enterica Serovar enteritidis is a desirable aim of vaccination. Live attenuated vaccines are widely used in the poultry industry, breeding and layers stock. Using this type of vaccines has some disadvantages, mainly because there is a small risk that attenuated forms of a pathogen can revert to a dangerous form, and, in addition, the pathogen may infect other animals. A vaccine not containing any live form of the pathogen which will stimulate the intestinal immune response can be of enormous advantage to the poultry industry.
There is a need for routes to administer vaccines to aquatic farm as well as terrestrial animals that are not costly and do not require laborious efforts. Oral administration of vaccines will be ideal for these purposes. Oral delivery of vaccines to humans is also desirable, as this mode of administration does not require professional manpower and prevents the discomfort involved in parenteral administration. For successful oral delivery, antigens should be protected from chemical and enzymatic degradation that may occur during processing the antigens into food or a feed composition and through the delivery via the animal or human gastrointestinal tract. In addition, the antigen should overcome structural barriers that preclude entry into the animal or access to the target destination.
Microalgae (single cell alga or phytoplankton) represent the largest, but most poorly understood, kingdom of microorganisms on the earth. Like plants are to terrestrial animals, the microalgae represent the natural nutritional base and primary source of all the phytonutrients in the aquatic food chain. The use of algae for vaccine production offers several advantages such as low cost, safety and easy scaling up.
Expression of recombinant proteins in algae has been reported, and various methods are available for production of exogenous proteins within the algae cells, particularly within the cell plastid. International (PCT) Application Publication No. WO 2011/063284 discloses methods of expressing therapeutic proteins in photosynthetic organisms, including prokaryotes such as cyanobacteria, and eukaryotes such as alga and plants. Transformation of eukaryotes is preferably into the plastid genome, typically into the chloroplast genome.
U.S. Pat. Nos. 7,410,637 and 8,282,915 disclose delivery systems and methods for delivering a biologically active protein to a host animal. The systems and methods provided include obtaining an algal cell transformed by an expression vector, the expression vector comprising a nucleotide sequence coding for the biologically active protein, operably linked to a promoter. In one illustrated embodiment, the biologically active protein is an antigenic epitope and upon administration to the animal the algal cell induces an immune response in the host animal.
International (PCT) Application Publication No. WO 2002/076391 discloses the use of microbial cells which are used as feed components in aquaculture or agriculture, and which also contain exogenous peptides, proteins, and/or antibodies, which will convey resistance or immunity to viral or bacterial pathogens or otherwise improve the health and performance of the species consuming said microbial cells. The microbial cells can be yeast, fungi, bacteria, or algae. The proteins and/or antibodies may be expressed inside the microbial cells by direct genetic modification of the microbe itself, or by the infection of the microbe with a virus that has been altered to express the protein of interest.
International (PCT) Application Publication No. WO 2008/027235 discloses methods for prevention, amelioration or treatment of a disease or disorder in an aquatic animal, by feeding the aquatic animal directly or indirectly with genetically modified microalgae that express a recombinant molecule that specifically targets one or more key epitopes of a pathogen that infects the aquatic animal.
U.S. Patent Application Publication No. 2011/0014708 discloses method of producing a foreign desired gene product in algae that comprises weakening or removing the algae cell wall by a protein enzyme solution to facilitate the gene transfer and a feed composition comprising the transgenic algae or its offspring. The invention also provides a modified nucleic acid for expressing bovine lactoferricin (LFB) in algae.
International (PCT) Application Publication No. WO 2014/030165 to the inventor of the present invention, published after the priority date of the present invention, discloses transgenic microalgae expressing exogenous biologically active proteins and use thereof for oral delivery of the biologically active proteins to animals and humans.
However, there is still an unmet need for and it would be highly advantageous to have an effective oral delivery system for vaccination that is easy for production and use, maintains the immunogenic activity of the antigen and facilitates absorption of the antigen by an organism.